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Cell signaling through O-GlcNAc reader proteins

通过 O-GlcNAc 读取蛋白的细胞信号传导

基本信息

批准号：
10656649
负责人：
MICHAEL S BOYCE
金额：
$ 32.04万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10656649
关键词：
14-3-3 Family Ablation Binding Binding Proteins Biochemical Biological Biological Process Biophysics Cell model Cell physiology Cells Comparative Study Complex Cytoplasmic Protein Cytoskeletal Filaments Cytoskeletal Proteins Deubiquitination Diabetes Mellitus Diffusion Disease Enzymes Future Genetic Glycoproteins Goals Homo Human Importins In Vitro Individual Intermediate Filament Proteins Intermediate Filaments Link Malignant Neoplasms Mammalian Cell Mammals Mediating Methods Modeling Multiprotein Complexes Mus Nerve Degeneration Nuclear Nuclear Envelope Nuclear Import Nuclear Pore Nuclear Pore Complex Proteins Nuclear Proteins Organelles Peptides Phenotype Phosphoproteins Phosphorylation Physiological Polysaccharides Post-Translational Protein Processing Process Proteins Reader Regulation Reporting Resolution Role Signal Transduction Structure Testing Vimentin Work X-linked intellectual disability chromatin remodeling comparative glycosylation human disease inorganic phosphate insight neurofilament novel novel therapeutics nucleocytoplasmic transport paralogous gene protein complex protein function protein protein interaction receptor receptor binding sugar trafficking transmission process ubiquitin-protein ligase

项目摘要

Project Summary/Abstract O-linked b-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification (PTM) in mammals, decorating thousands of nuclear and cytoplasmic proteins. O-GlcNAc cycling is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases, such as cancer, diabetes and neurodegeneration. Despite this broad pathophysiological significance, major aspects of O-GlcNAc signaling remain incompletely understood, including the biochemical mechanisms through which O-GlcNAc transduces information. At the start of our project, several studies had shown that O-GlcNAcylation mediates protein-protein interactions (PPIs) in processes as diverse as chromatin remodeling, deubiquitination and nuclear envelope assembly. These results suggested that O-GlcNAc might signal through conserved modes of PPI. However, little was known about either the structure or function of these intracellular glycoprotein-protein complexes. We hypothesized that mammalian “reader” proteins might exist and transduce O-GlcNAc signals. In the first project period, we devised and deployed a method to identify proteins that bind specifically and directly to O- GlcNAcylated peptides and proteins in vitro and in human cells. These include the 14-3-3 family, which are well- known to bind to phosphoproteins. Our results indicate that 14-3-3 proteins may be signal integrators, mediating the extensively documented crosstalk between O-GlcNAc and O-phosphate, with broad implications for intracellular signaling. In addition, we identified and characterized O-GlcNAc-mediated PPIs among nuclear import receptors, intermediate filament (IF) cytoskeleton proteins and ubiquitin E3 ligase complex components. Building on these results, we will study a focused set of diverse model O-GlcNAc-binding proteins in the next project period. This strategy will generate new insight into the functions of individual O-GlcNAc-mediated PPIs in particular cell biological processes, while also providing a comparative perspective across reader proteins, allowing us to discern general themes of O-GlcNAc-mediated PPIs at the biophysical, biochemical and physiological levels. The long-term objective of our work is to understand the principles, mechanisms and functions of O- GlcNAc-mediated PPIs in mammals. In the next project period, we will advance this goal through three Specific Aims. In Aim 1, we will define the biochemical scope and phenotypic effects of O-GlcNAc binding by 14-3-3 proteins. In Aim 2, we will determine the role of O-GlcNAc/nuclear transport receptor interactions in inter-organelle trafficking. In Aim 3, we will dissect the functional importance of O-GlcNAc-mediated interactions in two model IF proteins. We believe our proposed work will significantly advance the field of intracellular signaling both by elucidating the mechanisms and functions of individual O-GlcNAc-mediated PPIs and by revealing conserved biochemical principles of O-GlcNAc recognition across reader proteins.

项目摘要/摘要 O-连接的b-N-乙酰氨基葡萄糖(O-GlcNAc)是一种普遍存在的翻译后修饰(PTM) 哺乳动物，装饰着成千上万的核蛋白和细胞质蛋白。O-GlcNAc循环是一个重要的调节因素细胞生理学的许多方面，在许多人类疾病中调节失调，如癌症、糖尿病和神经退化。尽管具有广泛的病理生理意义，O-GlcNAc信号的主要方面仍然不完全清楚，包括O-GlcNAc转导的生化机制信息。在我们项目开始的时候，几项研究已经表明O-GlcN酰化是蛋白质-蛋白质的中介染色质重塑、去泛素化和核膜等过程中的相互作用(PPI) 集合。这些结果表明O-GlcNAc可能通过PPI的保守模式来传递信号。然而，几乎没有对这些细胞内糖蛋白-蛋白质复合体的结构或功能都是已知的。我们假设哺乳动物的“阅读器”蛋白可能存在，并转导O-GlcNAc信号。在在第一个项目期间，我们设计并部署了一种方法来识别特异性和直接与O-结合的蛋白质体外和人体细胞中的GlcNacyl化多肽和蛋白质。这些人包括14-3-3家庭，他们很好- 已知可与磷蛋白结合。我们的结果表明，14-3-3蛋白可能是信号整合因子，介导广泛记录的O-GlcNAc和O-磷酸之间的串扰，具有广泛的意义细胞内信号。此外，我们在核进口中鉴定和表征了O-GlcNAc介导的PPI 受体、中间丝(IF)细胞骨架蛋白和泛素E3连接酶复合体。建房在这些结果的基础上，我们将在下一个项目期间研究一组不同模型的O-GlcNAc结合蛋白。这一策略将对单个O-GlcNAc介导的特定细胞PPI的功能产生新的见解生物过程，同时也提供了读者蛋白质的比较视角，使我们能够识别 O-GlcNAc介导的PPI在生物物理、生化和生理水平上的一般主题。我们工作的长期目标是了解O-O的原理、机制和功能。哺乳动物中GlcNAc介导的PPI。在接下来的项目期间，我们将通过三个具体的项目来推进这一目标目标。在目标1中，我们将定义14-3-3与O-GlcNAc结合的生化范围和表型效应蛋白质。在目标2中，我们将确定O-GlcNAc/核转运受体相互作用在细胞器间的作用贩卖人口。在目标3中，我们将在两个模型IF中剖析O-GlcNAc介导相互作用的功能重要性蛋白质。我们相信，我们提议的工作将显著推动细胞内信号领域的发展，阐明O-GlcNAc介导的单个PPI的机制和功能并揭示保守的 O-GlcNAc跨阅读蛋白识别的生化原理。